The present invention relates to methods of generating glia cells such as oligodendrocyte like cells and the use of same for the treatment of medical conditions of the CNS.
Myelin, the fatty substance which encloses certain axons and nerve fibers, provides essential insulation, and enables the conductivity of nerve cells which transmit electrical messages to and from the brain.
Aberration in myelination can lead to several pathologies in the central nervous system. These include for example, autoimmune diseases (e.g Multiple Sclerosis), congenital leukodystrophies (e.g Pelizaeus-Merzbacher, vanishing white matter, adrenoleukodystrophy), infectious diseases (e.g. progressive multifocal leukoencephalopathy, postinflammatory demyelinated lesions), neurodegenerative diseases (e.g. multisystem degeneration), vascular diseases (vascular leukoencephalopathies, subcortical infarcts), congenital genetic defects (e.g. amyotrophic lateral sclerosis [ALS], Alzheimer disease, Parkinson disease) and brain and spinal cord trauma or injuries which are demyelinative and possibly neoplasms (e.g. oligodendrio-glioma).
Treatment of these pathologies may be effected by cell replacement therapies which eventually may allow achieving regional or even global repair of myelin as indicated by experiments in MBP-deficient shiverer mice (e.g., Yandava et al., 1999).
Oligodendrocytes extend as many as 50 processes which wrap around axons to form myelin sheaths. In vitro and in vivo, the development of oligodendrocytes proceeds in steps from neural stem cells to bipolar progenitors, then to multipolar precursor cells having several main processes which subsequently arborize and form the multiple branches of the mature cells (Rogister et al., 1999; Grinspan, 2002). Early oligodendrocytes precursors (OP) express the PDGF-receptor and later the specific O4 sulfatide marker of pre-oligodendrocytes which persists in ramified immature oligodendrocytes, then the maturating cells express galactocerebrosides (O1, GalC) and 2′,3′ cyclic nucleotide 3′ phosphodiesterase (CNP), and at the final stage become postmitotic mature oligodendrocytes that synthesize the myelin membrane components such as proteolipid protein (PLP) and myelin basic protein (MBP).
Embryonic stem (ES) cell lines being amenable to mass culture and differentiation into specific cell lineages in vitro are a potential large scale source of oligodendrocytes for brain and spinal cord transplantation, which has been studied in experimental models using mouse ES cells (Liu et al., 2000; Zhang et al., 2004; Glaser et al., 2005; Zhang et al., 2006). For successful transplantation, the cells need the capacity to migrate into the CNS and, therefore, the appropriate stage of cell differentiation must be defined, since more differentiated oligodendrocytes migrate minimally but conversely the cells that migrate most are less likely to differentiate into mature myelinating cells (Warrington et al., 1993; Foster et al., 1995; Yandava et al., 1999).
Zhang et al (2004; 2006) describe a procedure which converts murine ES cells into highly branched and ramified oligodendrocytes and produces progenitor which upon implantation in shiverer mouse brain tissue, migrate and form dense arrays of myelinated nerve fibers. However, the need for human ES cells which can be converted and expanded into functional oligodendrocytes is yet far from being fulfilled.
The availability of human ES cell (huESC) lines, derived from supernumerary human IVF blastocysts, provides a potential large scale source of human oligodendroglial and neural precursor cells that could be used in clinical settings to treat a variety of severe human neurological diseases, congenital or acquired. Neural stem cells from brain or spine of aborted human fetuses may be another source of neural stem cells, but preparing engraftable quantities of cells from fetal or adult human brain presents many problems (Goldman, 2005). While human fetal brain cells can differentiate into ramified mature oligodendrocytes (Zhang et al., 2000), most studies show that fetal brain precursor cells lose their ability to produce oligodendrocytes upon expansion (Chandran and Compston, 2005). Therefore, the use of laboratory-established huESC lines that can be expanded in relatively large scale cultures, would be highly advantageous to prepare the grafts.
Previous attempts to use human ES cells lines for deriving oligodendrocytes have yielded only partial success. A first study described the preparation of neural tube-like rosettes which were expanded by bFGF into neurospheres and eventually transplanted into the third ventricule of newborn mice brain (Zhang et al., 2001). In this work only elongated bipolar O4+ OPC were obtained and no myelination was shown following transplantation. Another study (Reubinoff et al., 2001) produced neurospheres from huESC with EGF and bFGF, and showed multipolar O4+ cells with a few processes, as well as CNP-positive cells after transplantation but without evidence for myelination. A further study, with huESC-derived neurospheres that were produced with bFGF and noggin yielded O4+ precursors but no ramified or mature cells, and no transplantation was done (Itsykson et al., 2005).
The inability of neurons in the mammalian central nervous system (CNS) to regenerate axons is due to inhibitory influences by glial cells of the CNS, which prevent the re-activation of growth promoting genes. However, axonal regeneration occurs in the peripheral nervous system (PNS). Retinoic acid-mediated signal transduction is known to induce these regenerative processes in the PNS. Retinoic acid (RA) is one of the active forms of vitamin A and is involved in life maintaining processes such as reproduction, embryonic development, vision, growth, cellular differentiation and proliferation, tissue maintenance and lipid metabolism. By activating a number of regulatory genes and signaling molecules, retinoic acid plays a crucial role during the development of the vertebrate nervous system. RA also initiates cell development of immature blood cells. One of the many medical uses of RA is for the treatment of Acute Myeloid Leukemia. Administration of RA causes the highly proliferative immature blood cells typical of this disease, to differentiate and develop into functional cells.
Because of its known functions, retinoic acid was used to induce neural differentiation and neurospheres in murine ES cells (Bain et al., 1995; Liu et al., 2000). Following this approach (Nistor et al., 2005), retinoic acid was used for the induction of neural-lineage cells from HuESC, in concurrence with preferential selection of oligodendrocyte-lineage cells by media components and matrigel adherence. Although “high purity functional oligodendrocytes from huESC” were claimed, no well differentiated, highly branched and ramified oligodendrocytes were produced, and after transplantation to spinal cord of shiverer mouse, there were only small patches of MBP-positive cells with no evidence for extended areas of remyelination and no myelinated nerve fibers of at least 100 μm.
All the described attempts to produce fully differentiated and mature oligodendrocytes from human ES cells convey that procedures which have been successful with murine ES cells cannot be extrapolated to human ES cells. Thus, the need for production of fully differentiated and mature human oligodendrocytes still exists.
For this purpose, key genes, encoding transcription factors which are obligatory for the development of the oligodendrocyte lineage need to be studied, in order to define the agents that are needed to convert huESC into oligodendrocytes, and the agents which delay this process, and need to be inhibited. For example, Bone Morphogenetic Proteins (BMPs) are group of growth factors known for their ability to induce the formation of bone and cartilage. Signal transduction through BMPs, is important for the development of the heart, central nervous system, and cartilage, as well as post-natal bone development. Noggin, which binds to members of the TGF-β superfamily, plays a crucial role in bone development and neurulation, by regulating the functions of BMP's. For example, it was found that noggin counteracts the effect of BMPs, which in turn were found to inhibit oligodendrocyte development from rat fetal brain (Mehler et al., 1997; Mehler et al., 2000).
There is thus a widely recognized need for producing fully differentiated, and mature oligodendrocytes propagated and expanded from human ES cells, that addresses these deficiencies.